RESEARCH ARTICLE 2383
Development 135, 2383-2390 (2008) doi:10.1242/dev.023275
Association of trxG and PcG proteins with the bxd
maintenance element depends on transcriptional activity
Svetlana Petruk1, Sheryl T. Smith2, Yurii Sedkov1 and Alexander Mazo1,*
Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of
expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes
can antagonize each other’s function, whereas mutations of genes within each group have synergistic effects. Here, we show in
Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of
the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland
with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and
Asx require Trithorax in order to bind to their target genes. Together, our data argue that at the single-cell level, association of
repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or
antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically.
INTRODUCTION
Two large gene families, the trithorax group (trxG) and the
Polycomb group (PcG), are required to maintain the appropriate
state of Hox gene expression throughout development (reviewed by
Grimaud et al., 2006). The traditional view is that trxG proteins are
required to maintain the active state of Hox gene expression,
whereas PcG proteins repress their expression. Combining
mutations of members of the same group enhances their mutant
phenotype, whereas combining mutations from each group results
in mutual suppression. Despite this general trend, some trxG genes
can act as both activators and repressors (Busturia et al., 2001;
Hodgson et al., 2001; Horard et al., 2000). Mutations in several PcG
genes can enhance the phenotypes of trxG mutants, suggesting that
a subset of PcG genes are required to activate as well as to suppress
Hox expression (Gildea et al., 2000; LaJeunesse and Shearn, 1996;
Milne et al., 1999; Sinclair et al., 1992). One such example is the
PcG gene Additional sex combs (Asx), which is required for both
activation and repression of different aspects of Hox expression in
Drosophila embryos (Milne et al., 1999). Thus, there are some trxG
and PcG proteins that have dual roles and, depending on a particular
context, may act as activators or repressors. It has been suggested
that these proteins should be called enhancers of trithorax and
Polycomb (ETPs) (Gildea et al., 2000).
Genetic experiments have suggested that PcG/trxG proteins might
alter the chromatin structure of their target genes (Grimaud et al.,
2006). Consistent with these genetically derived concepts,
accumulating data suggest that trxG and PcG proteins regulate
transcription of their target genes by altering their chromatin
structure. They are found in multiprotein complexes that either
modify histones within nucleosomes, or remodel chromatin, or are
components of the general transcriptional machinery. At present, we
know the composition and enzymatic activities of several trxG
1
Department of Biochemistry and Molecular Biology, Thomas Jefferson University,
Philadelphia, PA 19107, USA. 2Department of Biology, Arcadia University, Glenside,
PA 19038, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 16 May 2008
proteins and their complexes in Drosophila. The Trithorax (Trx)containing complex TAC1 possesses both histone acetyltransferase
(HAT) and histone H3 lysine 4 (H3-K4) methyltransferase
(HMTase) activities (Petruk et al., 2001; Smith et al., 2004). Another
trxG protein, Ash1, is also an HMTase. Ash1 was previously shown
to methylate a number of residues in histones H3 and H4 [H3-K4,
H3-K9 and H4-K20) (Beisel et al., 2002; Byrd and Shearn, 2003)],
but recent analysis suggests that it methylates exclusively H3-K36
(Tanaka et al., 2007). A trxG BRM complex is closely related to the
well-known yeast SWI/SNF ATP-dependent chromatin remodeling
complex (Papoulas et al., 1998). Several components of the BRM
complex, including Brahma (Brm), Moira (Mor) and Osa, are
encoded by trxG genes (Collins and Treisman, 2000; Crosby et al.,
1999; Papoulas et al., 1998). Although the trxG protein Kismet (Kis)
is similar to the SWI/SNF family of ATP-dependent remodeling
factors, it is a general factor at some stages of transcriptional
elongation (Daubresse et al., 1999; Srinivasan et al., 2005). Several
other molecularly characterized trxG proteins are thought to be
general transcription factors. For example, Skuld (Skd) and Kohtalo
(Kto) encode homologs of TRAP240 and TRAP230, two subunits
of the Drosophila Mediator complex (Janody et al., 2003). Two
purified PcG complexes, PRC1 and PRC2, are also involved in
chromatin alterations. PRC1 ubiquitylates histone H2A at lysine 119
(Wang, H. et al., 2004) and counteracts the chromatin remodeling
activity of the SWI/SNF complex (Francis et al., 2001). The PRC2,
or E(z)-Esc, complex may have several HMTase activities due to the
SET domain of Enhancer of Zeste [E(z)], the major one being
methylation of H3-K27 (Cao et al., 2002; Czermin et al., 2002;
Kuzmichev et al., 2002; Muller et al., 2002).
The targets of trxG and PcG proteins are not limited to the Hox
complexes. Both traditional cytological mapping of binding sites
on salivary gland polytene chromosomes (reviewed by Brock
and van Lohuizen, 2001) and recent genome-wide chromatin
immunoprecipitation (ChIP) assays (Negre et al., 2006; Schwartz et
al., 2006) demonstrate that these proteins are associated, often
jointly, with a very large number of genes. The maintenance of gene
expression by these groups of proteins is mediated by trxG and PcG
response elements (TREs and PREs, respectively). These elements
have been most extensively studied in the regulatory regions of the
DEVELOPMENT
KEY WORDS: Trithorax, Polycomb, Epigenetic regulation, Maintenance elements, Homeotic genes
2384 RESEARCH ARTICLE
Development 135 (14)
MATERIALS AND METHODS
Drosophila stocks
B1
2
5
1
2
3
The following trxG alleles were used: ash1 , brm , brm , skd , skd , kto ,
kis1, kis2, dev1, dev2, mor2, osa1, urd1, Df(urd), sls1, vtd (from J. Kennison,
NIH, Bethesda, MD); ash111, ash12, ash122, ash22, ash218 (from A. Shearn,
John Hopkins University, Baltimore, MD); Asx3 (from H. Brock, University
of British Columbia, Vancouver, Canada), kto1, osa2, mor1 (from the
Drosophila Stock Center, Bloomington, IN).
Genetic analysis
The strategy to determine bxd regulatory sequences that are
responsive to trxG mutations is described in the legend to Table 1
and by Tillib et al. (Tillib et al., 1999). Construction of the bxd
transgenes has been described previously (Tillib et al., 1999).
Induction of the trx RNAi line has been described previously (Petruk et
al., 2006).
Immunostaining of polytene chromosomes
Polytene chromosomes from third instar larvae were prepared and
immunostained as described previously (Tillib et al., 1999). The following
antibodies were used: Trx N1 (rat, dilution 1:20) (Tillib et al., 1999); Ash1
(rabbit, 1:150) (Rozovskaia et al., 1999); E(z) (rabbit, 1:25; from R. Jones,
Southern Methodist University, Dallas, TX); Ph (rabbit, 1:120), Pc (rabbit,
1:100) and Asx (sheep, 1:30) from H. Brock; Osa (mouse, 1:15), Kis (rabbit,
1:50) and Brm (rabbit, 1:75) from J. Tamkun (University of California, Santa
Cruz, CA).
RESULTS
To examine whether trxG genes other than trx also have response
elements in the bxd ME, we determined the effect of trxG mutations
on the expression of white in the adult eye under the control of the
bxd ME in a transgene. Mutations in genes required for activation
should reduce expression of white. We have shown previously that
tests of trx function at the endogenous bxd ME in embryos give
essentially the same results as those monitoring white expression in
the eye, establishing the validity of this assay for the tests used here
(Tillib et al., 1999). The results of these tests were entirely
reproducible with two wild-type N transgenic lines (Fig. 1B). With
the exception of sls, for which only one allele is available, we tested
several alleles of most of the trxG genes examined. We also tested
one Asx allele, to gain information with regard to this unusual ETP
gene. As shown in Fig. 1A,B and Table 1, expression of the white
marker gene is reduced in heterozygous mutants of nine newly
Fig. 1. Multiple trxG genes are essential for functioning of the
bxd TRE/PRE maintenance element. (A) The effects of ash122 and
Asx3 on the eye color of ΔC1 transgenic flies. (B) Map of trxG and PcG
response elements in the bxd region of Ubx based on the results of the
genetic white tests shown in Tables 1 and 2. Data from Table 1: genes
that interact or do not interact with the bxd N constructs are shown in
red and black, respectively, above the map. Data from Table 2: a
scheme of the ΔC constructs used in these experiments is shown at the
bottom. The B TRE has been deleted in the constructs because of
redundancy with the C TRE (Tillib et al., 1999). Mapped Trx, Asx, Ash1
and Brm response elements are in red. Previously mapped PcG response
elements are in green (Tillib et al., 1999).
DEVELOPMENT
Bithorax complex (BX-C). There are multiple TREs and PREs in
the 300 kb BX-C region, and these elements tend to localize in close
proximity to one another in regions termed maintenance elements
(MEs) (Chan et al., 1994; Chang et al., 1995; Chiang et al., 1995;
Fritsch et al., 1999; Hagstrom et al., 1996; Orlando et al., 1998;
Simon et al., 1993; Strutt et al., 1997; Tillib et al., 1999). The beststudied ME is localized in the bxd regulatory region of Ubx ~25 kb
upstream of the Ubx promoter. A number of PREs and Trx-regulated
TREs have been mapped to juxtaposed DNA sequences in a 3 kb bxd
ME (Tillib et al., 1999). This organization suggests that these
proteins could interact in complex ways at the ME.
Despite advances in studies of the PcG and trxG proteins, there is
little understanding of whether they act in concert at a ME. Even in
the bxd ME, mapping data for trxG proteins is limited to Trx and
Ash1 (Beisel et al., 2002; Papp and Muller, 2006; Petruk et al., 2007;
Tillib et al., 1999). It seems likely that many trxG proteins besides
Trx should act there, because Trx interacts directly with several trxG
proteins. For example, Trx interacts directly with Snr1 (RozenblattRosen et al., 1998), a component of the BRM complex (Papoulas et
al., 1998). Similarly, Trx and Ash1 can interact at the protein level
(Rozovskaia et al., 1999). They are associated with the same regions
of Ubx in vivo (Petruk et al., 2007; Petruk et al., 2006) and share
most of their target genes (Rozovskaia et al., 1999). There is also
very little data on whether trxG proteins are dependent on each other
for binding to their target genes. The only exception is the finding
that binding of Trx to its target genes is strongly affected in ash1
mutants (Kuzin et al., 1994). Another important uninvestigated issue
is how trxG functioning relates to that of the PcG and ETP proteins,
such as Asx. The answers to these questions might reveal whether
different complexes have similar functions in different places, act in
the same place with different functions, or have different functions
in different places.
In this work, we show that many trxG genes are required for
functioning of the bxd ME. Genetic experiments show that the
response elements for the ETP gene Asx and the trxG gene ash1
either coincide with or are juxtaposed to the response element of trx.
Consistent with this, binding of Ash1 and Asx to all their target
genes is completely dependent on Trx, suggesting that they function
cooperatively. Surprisingly, although the response elements of trx
and brm are also juxtaposed, the BRM complex does not require Trx
for its association with target genes. At the single-cell level, binding
of Trx and components of the two major PcG complexes, PRC1 and
PRC2, to the bxd-ME-containing transgene in vivo is mutually
exclusive. Binding of PRC1 and PRC2 to their target genes is
independent of Trx. Thus, our results indicate that although multiple
trxG and PcG proteins are required for functioning of the bxd ME,
many may function independently. Importantly, association of
activators and repressors with the bxd ME correlates with the
transcriptional status of the gene.
RESEARCH ARTICLE 2385
Associations of trxG and PcG proteins
Table 1. Responses of N construct transgenes to interactions
with trxG alleles
trxG allele
N1
N2
trxG allele
N1
N2
ash1B1
ash111
ash112
ash122
Asx3
ash22
ash218
trxB11
brm2
brm5
osa1
osa2
mor1
mor2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Df(urd)
urd1
skd1
skd2
kto1
kto2
sls1
kis1
kis2
dev1
dev2
vtd3
vtd5
+
+
+
+
+
+
+
–
–
–
–
–
–
+
+
+
+
+
+
+
–
–
–
–
–
–
trxG alleles were crossed to two transgenic lines carrying independent insertions of
the two N constructs containing the 3 kb bxd region [Fig. 1 and see Tillib et al. (Tillib
et al., 1999)]. Comparisons of eye color were made between progeny carrying either
N construct alone and those carrying N construct in the background of a given trxG
allele. The decrease in eye color expression caused by a decreased dosage of wildtype trxG protein was designated (+), and no change in expression was designated
(–).
tested trxG genes: brm, osa, mor, ash1, ash2, sls, urd, skd and kto.
Three trxG alleles did not show genetic interaction with the bxd ME:
kis, dev (btl – FlyBase) and vtd.
Interestingly, the bxd-ME-interacting alleles include ash1 and
three other genes that interact with Trx. They encode components of
the BRM complex: brm, osa and mor. Ash1 and Snr1 (a component
of BRM) directly interact with Trx (Rozovskaia et al., 1999;
Rozenblatt-Rosen et al., 1998), and Ash1 has also been shown to
genetically and physically interact with the TAC1 component dCBP
(Nejire – FlyBase) (Bantignies et al., 2000). It is notable that the Asx3
mutation also caused a decrease in expression of the white gene (Fig.
1A), suggesting that despite the ETP nature of this gene, it behaves
like a trxG gene in this assay. This is a potentially important finding
because Asx interacts directly with Trx (J. Hodgson, personal
communication). Together, these results suggest that these proteins
might interact directly with Trx at their target genes. Since this also
assumes that their elements would be located in close proximity to
each other, we chose ash1, Asx and brm alleles for more detailed
analysis.
Fine mapping of the ash1, Asx and brm response elements within
the central C module of the bxd element was performed as
described previously (Tillib et al., 1999), using multiple transgenic
fly lines with constructs in which the C1, C2 or C3 sub-elements of
the bxd ME were deleted (Fig. 1A,B). Table 2 shows that ash1 and
brm response elements reside within the C2 element, which is
juxtaposed to the C1 trx response element. About half of the ΔC3
lines did not respond to the ash1 mutations, suggesting that the
response element for ash1 detected in C2 might extend into the C3
element. Interestingly, the response elements for Asx and trx
coincide in the C1 DNA element (Fig. 1B, Table 2). Note that the
Asx analysis includes fewer transgenic lines, especially C2 lines, as
this analysis was performed at later stages of this work when some
of the original lines had been lost. Therefore, we cannot exclude the
possibility that the Asx response element might also extend into the
C2 region.
These results suggest a very complex organization of the bxd ME,
in which trxG and PcG proteins occupy either the same or
juxtaposed response elements. This raises the question of how the
functioning of this element is mediated by multiple trxG and PcG
proteins. This might occur by alternative binding of PcG versus trxG
proteins, or by the preferential action of one of the two groups of
simultaneously bound proteins. This can be best addressed by
examining the association of these proteins in a single cell, where
transcription of the Ubx transgene is either on or off. It is well
established that many ME-containing transgenes have a variegated
phenotype in the eye, i.e. that the white reporter gene in these
transgenes is expressed in only a subset of cells in the eye.
Consistent with this, we found that the white reporter gene in our
construct also shows differential expression in the salivary glands of
Table 2. Determination of ash1, brm and Asx response elements in the bxd region of Ubx
Transgenic line
trxB11
Asx3
ash122
ash112
brm2
brm5
C1 TRE: trx
ΔC1
ΔC1
ΔC1
ΔC1
ΔC1
ΔC1
1-11-42
1-11-51
1-11-17
1-11-61
1-11-11
1-11-10
–
–
–
–
–
–
–
–
–
–
NT
NT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
C2 PRE: Scm
ΔC2
ΔC2
ΔC2
ΔC2
ΔC2
14-7-42
14-7-8
14-7-3
14-7-6
14-7-7
+
+
+
+
+
+
NT
NT
NT
NT
–
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C3 PRE: Psc, Pcl, Scm, pho
ΔC3
ΔC3
ΔC3
ΔC3
ΔC3
2-3-18
2-3-13
2-22-02
2-3-84
2-22-85
+
+
+
+
+
+
+
+
NT
NT
+
+
+
–
–
+
–
+
–
+
+
+
+
+
+
+
+
+
+
+
The effects of trxG alleles trxB11, ash112, ash122, brm2, brm5 and the ETP allele Asx3 on expression of N and ΔC transgenes containing deletions of the TRE or PRE (see Fig. 1).
Adult flies from each line were crossed to each of the tested alleles and the eye color of the progeny was compared to the parent line. A decrease in eye color expression in
the progeny was designated (+), and no change in eye color expression designated (–) as suggestive of a deleted response element for a given trxG or ETP protein. The
transgenes containing the deleted C1 and C2 elements showed no further change in expression in the trx and Asx or ash1 and brm mutants, respectively, suggesting that
response elements for Trx and Asx reside in the C1 element, and that response elements for Ash1 and Brm reside in the C2 element. The response element for Ash1 may
extend into the C3 element.
NT, not tested.
*As previously identified (Tillib et al., 1999).
DEVELOPMENT
trxG alleles
Δ region C module
TRE/PREs*
2386 RESEARCH ARTICLE
Development 135 (14)
Fig. 2. In any one cell in a Drosophila salivary gland, trxG and
PcG proteins alternatively associate with the bxd ME.
(A) Variegating phenotypes of the white reporter gene of the 18-15
transgenic line in the salivary gland. DAPI staining of the same glands is
shown in the lower panels. (B) Binding of trxG proteins Trx, Asx, Ash1
and Kis to chromosome 3 of the salivary gland polytene chromosomes
of the wild type and 18-15 transgenic line. The site of insertion of the N
construct is at the very tip of chromosome 3 and is indicated by
arrowheads. Trx, Asx and Ash1, but not Kis, bind simultaneously to the
site of insertion of the N construct in ~50% of nuclei from the salivary
glands prepared from the same larvae. (C) PcG proteins Ph, Pc and E(z)
bind to the site of insertion of the N construct in those nuclei where Trx
protein is not associated with this site.
third instar larvae (Fig. 2A). Thus, tests for direct binding of trxG
and PcG proteins to the bxd ME transgene in salivary glands provide
a unique opportunity to address the above questions in individual
cells in vivo.
We examined the physical association of several trxG and PcG
proteins with the site of insertion of the 18-15 transgene that carries
the wild-type N construct shown in Fig. 1B (Tillib et al., 1999). We
found that Ash1 and Trx are always associated together at the site of
insertion of the N transgene (Fig. 2B). Remarkably, the Asx protein
was also found exclusively at the Trx-associated chromosomal sites
(Fig. 2B). Together with localization of the Asx and trx response
elements in the same region of the bxd ME, and the fact that
expression of the white transgene is decreased in Asx3 mutants (Fig.
1A, Table 2), these results suggest that at least in the larvae and the
adults, this ETP protein is functioning exclusively as a trxG protein
at the bxd ME in transgenes. The above results are also consistent
with direct physical interactions between Trx and Asx (J. Hodgson,
personal communication). Kis was not found at the site of insertion
of this transgene (Fig. 2B), consistent with the results of the genetic
white tests, which suggested that there is no kis response element in
the bxd ME (Fig. 1B, Table 1). Brm and Mor could not be examined
in these experiments because they are associated in wild-type
animals with the same region where the transgene is inserted, at the
tip of chromosome 3.
Two PcG proteins, Pc and Ph (Polyhomeotic), that have response
elements in the bxd ME (Fig. 1B) (Tillib et al., 1999), associate with
the N transgene in vivo (Fig. 2C). Similarly, E(z) protein is also
associated with the bxd ME, which is consistent with previous ChIP
analysis of larval imaginal discs (Cao et al., 2002; Papp and Muller,
2006). However, in sharp contrast with the above results, we found
that Trx and each of the three tested PcG proteins were not
simultaneously associated with the insertion site of the N transgene.
Roughly one half of the nuclei from the same salivary gland
contained Trx but not PcG proteins bound to the transgene, or vice
versa (Fig. 2C).
The alternative association of several trxG activators and several
PcG repressors with the N transgene in different subsets of
chromosomes from the same gland indicates that N transgenes may
be either activated or repressed, respectively, in different cells in
salivary glands. This is in line with the variegated expression of the
white transgene observed in salivary glands (Fig. 2A). To test this
directly, we examined whether RNA polymerase II (Pol II) is
associated with the transgene insertion site. Fig. 3A shows that an
activated form of Pol II that is phosphorylated at Ser5 is co-localized
with Trx but not with Pc at the alternative binding sites, suggesting
that Trx binding correlates with activation of this transgene. By
contrast, strong binding of Trx and H3-meK27 are mutually
exclusive (Fig. 3B; co-localization of H3-meK27 and PcG proteins
cannot be tested because these antibodies were raised in rabbits).
Since H3-meK27 is a PRC2-generated mark of repressed
transcription (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et
DEVELOPMENT
Fig. 3. Association of trxG or PcG proteins with the bxd ME in
individual cells correlates with an activated or repressed white
reporter gene, respectively. (A) Pol II phoshorylated at Ser5 is
associated with the site of insertion of the N construct (arrowheads) in
the same Drosophila salivary gland cells as Trx (left), but not Pc (right).
(B) H3-meK27 is not associated with the site of insertion of the N
construct in the same cells as Trx.
al., 2002; Muller et al., 2002), this result indicates that all tested
components of the PRC1 and PRC2 PcG complexes are associated
with the repressed transgene.
The trxG and PcG genes interact genetically. Combining
mutations of different trxG (or PcG) genes enhances their mutant
phenotype, whereas combining mutations of trxG with PcG genes
suppresses their mutant phenotypes. The finding of response
elements for multiple, interacting trxG and PcG proteins in close
proximity to each other (Fig. 1B), and the demonstration of mutually
exclusive binding of trxG and PcG proteins to the activated and
repressed ME (Figs 2, 3), suggest potential competition and
Fig. 4. Effect of trx RNAi on association of trxG and PcG proteins
with their target genes. Salivary gland polytene chromosomes were
prepared from wild-type third instar Drosophila larvae and from the line
expressing trx RNAi as described (Petruk et al., 2006). The overall
structure of these chromosomes is indistinguishable (Petruk et al.,
2006). (A) Trx is essential for association of Ash1 and Asx. Binding of
Trx (green), Ash1 (red, column 2) and Asx (red, column 3) is completely
abolished in the trx RNAi line, whereas binding of the control protein,
Ecdysone receptor (EcR, green) is unaffected (column 1). (B) Trx is not
essential for association of the trxG BRM complex and Kis. Components
of the BRM complex, Brm and Osa, and trxG protein Kis (red) bind to
their sites on polytene chromosomes in a line expressing trx RNAi. Trx
binding is in green. (C) Association of the PcG complexes PRC1 and
PRC2 is not affected by induction of trx RNAi. The intensities and the
number of bands for the components of PRC1, Pc and Ph, and the
component of the PRC2, E(z) (all in red), are not significantly affected in
the trx RNAi line.
RESEARCH ARTICLE 2387
synergism between the trxG and PcG proteins and between different
trxG proteins, respectively. However, with the exception of two
reports demonstrating hierarchical binding of several PcG proteins
to the bxd ME in larval imaginal discs (Wang, L. et al., 2004), and
that binding of Trx is strongly affected in ash1 mutant larvae (Kuzin
et al., 1994), there are no data with regard to the interdependencies
between and within these two antagonistic groups of proteins for
association with their common target genes. To address this, we
asked whether elimination of the Trx protein by expressing a trx
RNAi construct affected in vivo binding of trxG and PcG proteins
to their binding sites on the salivary gland polytene chromosomes.
Expression of trx RNAi was achieved using the Gal4-UAS system,
as described previously (Petruk et al., 2006), and results in the
efficient removal of the Trx protein from all of its binding sites on
polytene chromosomes (Fig. 4A). The structure of the polytene
chromosomes and binding of the unrelated protein, Ecdysone
receptor (EcR), were not affected when trx RNAi was expressed
(Fig. 4A) (Petruk et al., 2006).
Strikingly, binding of Ash1 was completely abrogated following
induction of trx RNAi (Fig. 4A). This suggests that Trx is essential
either for the recruitment or for stable association of Ash1 with all
of its binding elements in the genome. These results are consistent
with direct interaction of these proteins, and with the results that
show that Trx and Ash1 are associated jointly at the juxtaposed
response elements of the bxd ME (Rozovskaia et al., 1999) (Figs 1,
2 and Table 2). This is also consistent with the finding that Trx and
Ash1 bind in vivo to essentially the same regions of Ubx (Petruk et
al., 2007; Petruk et al., 2006). Since binding of Trx is also strongly
affected in the ash1 mutant larvae (Kuzin et al., 1994), this suggests
that this dependency is reciprocal. Taken together, these results
suggest that Trx and Ash1 are two interacting, mutually dependent
trxG proteins. It is, however, important to note that these proteins do
not appear to be components of the same protein complex.
In similar experiments, we tested the effect of trx mutation on
binding to polytene chromosomes of the ETP protein Asx. trx
mutation resulted in a complete loss of Asx binding to polytene
chromosomes (Fig. 4A). These results are consistent with those
above showing that response elements of trx and Asx reside in the
same small C1 DNA element of the bxd ME (Fig. 1A,B, Table 2), as
well as with the fact that Trx and Asx proteins interact directly (J.
Hodgson, personal communication). The results indicate that, like
Trx and Ash1, Trx and Asx are intimately related in their functioning
at Ubx and other common target genes. Together, our results suggest
that these three proteins, Trx, Ash1 and Asx, might be involved in
direct interactions on the bxd ME.
By contrast, we were not able to detect any significant differences
in association of Kis, Brm and Osa with salivary gland polytene
chromosomes in the trx RNAi line (Fig. 4B). This is consistent with
the absence of Kis response elements in the bxd ME (Fig. 1B, Table
1), and suggests that Kis might not be directly involved in the
functioning of the epigenetic MEs, at least in the salivary glands.
However, the results for Brm and Osa, as components of the BRM
complex that are associated in close proximity to Trx on the bxd ME
(Fig. 1B, Table 2) and can genetically and physically interact with
TAC1, are very surprising. They imply that this complex functions
completely independently of TAC1. They also suggest that there is
no overall cooperativity in the association of trxG proteins with the
bxd ME, and that only a subset of trxG proteins is recruited to this
element synergistically.
We did not find significant differences in the association of the
components of the two major PcG complexes, PRC1 (Pc and Ph)
and PRC2 [E(z)], with their sites on polytene chromosomes in the
DEVELOPMENT
Associations of trxG and PcG proteins
trx RNAi line (Fig. 4C). Since binding of Ash1 and Asx is strongly
affected (Fig. 4A), these results also imply that PcG proteins
function independently of Ash1 and Asx. We did not detect any
increase in the number or intensity of the Pc and Ph polytene bands
in the trx mutant larvae, suggesting that removing trxG proteins
from their binding sites does not necessarily lead to enhanced
binding of the PcG proteins. It is therefore likely that there is no
continuous direct competition between these two groups of
opposing regulators for binding to their neighboring response
elements.
DISCUSSION
Despite much interest, there is little understanding of how the
epigenetic TRE/PRE-containing MEs function. One key
unresolved issue pertains to the organization of these complex
transcription regulatory elements with regard to the response
elements/binding sites of particular trxG and PcG proteins.
Response elements for several PcG proteins were mapped in the
bxd ME previously (Tillib et al., 1999), and some PcG proteins
were detected at this DNA element in ChIP assays (Cao et al., 2002;
Papp and Muller, 2006). However, information about the
association of trxG proteins in the bxd ME is very limited. We
previously mapped several Trx-dependent TREs in the bxd ME
(Tillib et al., 1999). In addition, we and others have detected Trx
and Ash1 proteins at the bxd ME in ChIP assays (Papp and Muller,
2006; Petruk et al., 2007; Petruk et al., 2006). Given the apparent
functional heterogeneity of the trxG proteins, it is revealing that
besides Trx, many other trxG genes are essential for functioning of
the bxd ME. Two of the interacting genes, skd and kto, encode
components of the Drosophila Mediator complex (Janody et al.,
2003), so it is possible that their role in the functioning of the bxd
ME relates to the transcription of some of the non-coding RNAs
that are known to be transcribed through this element [Petruk et al.
(Petruk et al., 2006) and references therein]. Ash2 is a component
of several purified MLL (a human homolog of Trx) protein
complexes (Dou et al., 2005; Nakamura et al., 2002; Yokoyama et
al., 2004). The identification of an ash2 response element in the bxd
ME suggests that a second putative Trx-containing MLL-like
complex might reside at the bxd ME. The genes urd and sls have
only been minimally characterized, mainly as suppressors of Pc
phenotypes. Therefore, it is premature to speculate about their
function at this element, although they clearly interact there in some
capacity.
Identification of multiple TREs and PREs within the same ME
raises an important question with regard to potential
interdependency or competition in the association of proteins from
the same and different protein families. To address this, we focused
on the fine mapping of response elements for several major trxG
genes that are essential for functioning of the bxd ME: ash1, the brm
component of the BRM chromatin remodeling complex, and the
ETP gene Asx. These proteins or components of their protein
complexes (i.e. Snr1, a component of BRM) can physically
associate with Trx (Rozenblatt-Rosen et al., 1998; Rozovskaia et al.,
1999) (J. Hodgson, personal communication). Thus, finding their
response elements either in DNA fragments that are juxtaposed to
(brm and ash1) or the same as (Asx) the previously mapped trx
response element is consistent with direct interactions of these
proteins with Trx. It should be noted, however, that all these proteins
are components of protein complexes other than the Trx complex
TAC1 (Papoulas et al., 1998; Petruk et al., 2001). Nevertheless, this
suggests that there might be interdependency in recruitment and/or
association of these protein complexes at the bxd ME. However, our
Development 135 (14)
results indicate that this suggestion is only partially true. Binding of
the components of the BRM complex and of another trxG protein,
Kis, were not affected by elimination of Trx. However, the
association of Ash1 and Asx at all their sites on the salivary gland
polytene chromosomes is completely dependent on the presence of
Trx. Previous results of the reciprocal experiments indicated that
binding of Trx is strongly decreased in ash1 mutant animals (Kuzin
et al., 1994). This suggests that Trx, Ash1 and Asx represent a
special, and at least partially interdependent, set of trxG proteins.
This also suggests, in contrast to the previously mentioned genetic
studies, that not all trxG proteins are mutually dependent in their
functioning.
Close proximity or even overlap between some TREs and PREs
in the bxd ME suggests the existence of potential competitive
relationships with regard to the binding of these functionally
opposing groups of proteins. Furthermore, some ChIP assays
indicate that some trxG and PcG proteins can bind to the bxd ME of
both the activated and silenced gene (Papp and Muller, 2006),
suggesting a potential interaction of these proteins on DNA. We
tested this by asking whether binding of the components of two
major PcG complexes, PRC1 and PRC2, is affected by elimination
of Trx. We did not detect any significant change in the number or
intensity of immunostained bands for all tested PcG proteins on the
polytene chromosomes of trx mutant larvae. This suggests that not
only is the association of PcG proteins independent of Trx, but also
that Trx is not essential for preventing binding of the PcG proteins
to their response elements. This is an important conclusion because
some genetic studies have proposed that the main function of Trx
and Ash1 is to prevent silencing by the PcG proteins (Klymenko and
Muller, 2004).
An important issue in understanding the molecular mechanism of
trxG/PcG functioning is to correlate their association at MEs with
the state of expression of their target genes. Although most of the
existing data were obtained in cultured cells, two studies addressed
this issue in Drosophila larval tissues. ChIP analysis in larval
imaginal discs suggests that some trxG and PcG proteins are
associated with the bxd ME irrespective of the status of gene
expression (Papp and Muller, 2006). However, the results of another
study suggest alternative association of Trx and Pc at the site of the
endogenous BX-C on polytene chromosomes from both fat body
and salivary glands, where BX-C is correspondingly activated or
repressed (Marchetti et al., 2003). Ideally, to resolve this issue it is
essential to investigate the association of PcG and trxG proteins with
the ME in the same tissue at the single-cell level and at a gene of
defined expression status. We established such a test system in
which the bxd-ME-containing transgene is either activated or
repressed in cells within the same salivary gland. Direct
visualization of the association of different proteins to the site of
insertion of this transgene clearly indicates that major trxG and PcG
proteins bind to the bxd ME in an alternative fashion. Importantly,
using markers for activated and repressed transcription, we were
able to correlate binding of trxG and PcG proteins in a single cell
with either the activated or repressed bxd transgene, respectively.
The differences between our results and those of Papp and Mueller
(Papp and Muller, 2006) might be explained by technical differences
and by the fact that trxG and PcG proteins may behave differently
in different tissues and/or in polyploid versus diploid cells. It is
important to note that although our analysis is limited to studies of
a transgene, the detected alternative association of Trx and Pc on the
bxd ME transgene correlates well with the results obtained at the
endogenous BX-C on polytene chromosomes (Marchetti et al.,
2003). We conclude, therefore, that on a cell-by-cell basis, binding
DEVELOPMENT
2388 RESEARCH ARTICLE
of trxG and PcG proteins is strictly dependent on the status of gene
expression, in that they bind alternatively to the epigenetic
regulatory elements of either activated or repressed target genes,
respectively.
In summary, this is the first work on the fine mapping of multiple
TREs at any target gene. This is also the first assessment of mutual
dependencies within the trxG group of activators and between the
trxG and PcG of antagonistic proteins. It provides a glance of the
enormously complex regulatory element that binds proteins with
opposite transcriptional regulatory activities. The main conclusions
of this study are that two major trxG proteins, Trx and Ash1, and the
ETP protein Asx, constitute a specific subgroup of interacting
proteins that depend on each other in their functioning at the bxd ME
and throughout the genome. Although multiple trxG proteins are
essential for epigenetic functioning of the bxd ME, their association
with this element and other binding sites in the genome might not
necessarily require Trx and associated proteins, as exemplified by
the components of the BRM complex and Kis. The components of
the major PcG complexes, PRC1 and PRC2, also associate with
target genes independently of Trx, Ash1 and Asx. Another important
conclusion of this work is that trxG and PcG proteins are associated
with the bxd ME only at activated and repressed genes, respectively.
It will be important to determine whether the choice between the
establishment of trxG-mediated activation or PcG-mediated
repression occurs only at very specific early stages of development,
or whether it can also occur at later developmental stages.
We thank H. Brock, J. Hodgson and J. Jaynes for critical comments on the
manuscript, J. Tamkun, R. Jones and H. Brock for antibodies and J. Kennison,
A. Shearn and H. Brock for mutant stocks. This work was supported by grants
NIH 1R01GM075141 and March of Dimes 6-FY06-346 to A.M.
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